Bacterial resistance to antimicrobial agents has increased in recent years and now represents a significant threat to successful antibiotic therapy. One example of this phenomenon is the development of resistance to B-Iactam antibiotics. B-lactam antibiotics, such as the penicillins and cephalosporins, are among the most frequently used antimicrobial agents. The most common mechanism of resistance to B-lactam antibiotics is the production of B-lactamases, which cleave the antibiotic, rendering it harmless to bacteria. Based on primary sequence homology, B-lactamases have been grouped into four classes. Classes A, C and D are active-site serine enzymes that catalyze, via a serine-bound acyl-enzyme intermediate, the hydrolysis of the B-Iactam antibiotic. Class B enzymes require zinc for activity and catalysis does not proceed via a covalent intermediate. Because of the diverse range of substrate specificities of these enzymes, virtually all B-lactam antibiotics are susceptible to hydrolysis. Clearly, the design of new antibiotics that escape hydrolysis by the growing collection of B-lactamase activities will be a challenge. It will be necessary to understand the catalytic mechanism and basis for substrate specificity of each class of B-lactamase. The goal of this work is understand how the amino acid sequence determines the structure, catalysis, and substrate specificity of the IMP-I B-lactamase of class B and the P99 class C B-lactamase. This will be achieved by randomizing amino acid positions in the active-site pocket of each enzyme to sample all possible amino acid substitutions. All of the random substitutions will then be screened to identify those substitutions that alter the substrate specificity of the enzyme. Enzymes containing substitutions that alter substrate specificity will be purified and characterized biochemically. The sets of random substitutions will also be screened using phage display methodology to identify residues critical for catalysis. A further goal of this proposal is to use the detailed knowledge of the interface between B-lactamase inhibitory protein (BLIP) and B-lactamase, in combination with random mutagenesis and phage display, to create derivatives of BLIP that bind and inhibit B-lactamases and penicillin binding proteins. The new' BLIP derivatives will be characterized biochemically and structurally. The information gained from these studies will be useful for the rational design of new antibiotics and inhibitors.